Immunologic and physicochemical studies of Bermuda grass pollen antigen BG60

Abstract

BACKGROUND: In a previous study we showed that antigen BG60 of Bermuda grass pollen contains isoallergens. Because the yield of purified isoallergens was low when a chromatofocusing technique was used, it was difficult to carry out further studies, such as determination of carbohydrate composition and structure. OBJECTIVE: The aim of this study was to establish a procedure to purify antigen BG60 proteins as a group and to characterize this group’s physicochemical and immunologic properties. METHODS: A combination of chromatographic techniques (ion-exchange, gel filtration, blue gel affinity, and reverse-phase high-performance liquid chromatography) was used for the purification of BG60. Immunoblot and ELISA techniques were used to study BG60-specific IgE and IgG antibodies in patients’ sera. The role of the carbohydrate moiety in antigenicity and allergenicity was examined with monoclonal antibodies and allergic sera by using periodate-treated BG60. Its carbohydrate composition was analyzed by high-performance anion-exchange chromatography with a pulsed amperometric detector. RESULTS: Homogeneity of BG60 was demonstrated by a single sharp peak in reverse-phase high-performance liquid chromatography, a single band in sodium dodecylsulfate–polyacrylamide gel electrophoresis, and only one band stained by anti-BG60 monoclonal antibody. BG60-specific IgE and IgG antibodies were shown to be present in allergic sera. Six plant lectins were found to react with BG60. On periodate treatment, BG60 reduced binding toward its monoclonal antibody and human IgE and IgG. Carbohydrate composition analysis showed that BG60 contains three kinds of sugars: mannose, N-acetylglucosamine, and fucose (in a ratio of approximately 3:2:1) and a minute amount of xylose. The carbohydrate content is approximately 7.5%, and peptide content is about 92.5%. CONCLUSION: A procedure was established for the purification of a large quantity of the BG60 antigen. The results suggest that the carbohydrate moiety of antigen BG60 may play an important role in the immune response. ( J Allergy Clin Immunol 1996;98:486-94.)